Development of Ribozyme P in vitro system for Acinetobacter baumannii

DAVIES SALA, C.; ABDIAN, P.; ZORREGUIETA, A; TOLMASKY, M E

Mar del Plata

Congreso; IX Congreso Argentino de Microbiología General, SAMIGE; 2014

Asociación Argentina de Microbiología General

Acinetobacter baumanni ATCC 17978 protein subunit was amplified by PCR and cloned in a  pET22b+ expression vector under T7 promoter carrying six Histidine repeats in its C-terminal. Protein subunit was then over-expressed in E. coli BL21(DE3) and purified by ultracentrifugation schedule followed by a nickel column chromatography. RNA subunit sequence in A. baumanni ATCC 17978 was determined by genome sequence analysis and comparison. The predicted DNA sequence was amplified by PCR and cloned in pBBR1-MCS2 under the control a the T7 promoter. The RNA subunit of Ribozyme P was obtained by in vitro transcription using the recombinant plasmid as template . Functionality of the holoenzyme will be tested using as a substrate a pre-tRNA (the natural substrate of the RNase) or a target mRNA and an EGS, whose activity to mediate RNase P RNA cleavage has been proven. We believe that the development of the A. baumanni RNase P in vitro system will contribute to increase antisense technology tools in a clinically relevant bacterium as well as basic knowledge on the ribozyme. The A. baumanni RNase P in vitro system will be used to analyze the ability of EGSs to induce cleavage of a relevant target RNA in a clinically relevant bacterium.